Heme, Ubiquitin and Lung Cancer

Uncover how the heme-oxidative stress pathway controls protein degradation via the Ubiquitin-Proteasome System

Ubiquitin-Proteasome System (UPS)-regulated protein degradation is an irreversible mechanism utilized by numerous biological processes that are modulated through selective elimination of proteins, therefore making the UPS the ultimate on–off switch of a cell. The ubiquitylation process is controlled through an enzymatic cascade in which molecules of ubiquitin are acti­vated by an E1 enzyme, transferred to an E2 ubiquitin-conjugating enzyme and finally transferred to a protein-substrate recognized by an E3 ubiquitin ligase.  In humans, there are only two E1 enzymes, ~30 E2 enzymes, and ~600 E3 enzymes. This remarkable evolutionary feature of the UPS cascade allows the cells to direct the ubiquitylation process with extreme selectivity and specificity. It is estimated that >80% of proteins undergo UPS-mediated degra­dation, therefore the selection of specific substrates by E3 ubiq­uitin ligases in response to specific stimuli plays a central role in the life of the cells.
 
Research from our and other laboratories uncovered that binding of signaling heme to several proteins, such as BACH1 or p53, promotes their UPS-dependent degradation, thus revealing unprecedented roles of signaling heme in regulating protein turnover via the UPS. Interestingly, despite the potential widespread impact of the heme-UPS pathway on the mechanisms controlling cellular homeostasis, heme-dependent protein degradation represents a fundamental and complex scientific question that remains poorly studied. To address this question our laboratory leverages a broad spectrum of experimental systems to:
 
1.  Define the heme degradome (i.e., heme-regulated proteome).

We seek to identify new heme-UPS substrates and characterize their downstream pathways. We also investigate the molecular machineries regulating the heme degradome, such as E3 ubiquitin ligases and deubiquitylating enzymes, that may work as heme sensors by coupling regulation of protein degradation with variations of the heme signal. 
 
2.  Unravel the mechanisms underlying heme-regulated protein destruction. 

Our studies demonstrate a novel regulatory role for heme in promoting the interaction between a ubiquitin ligase (i.e., FBXO22) and its substrates (i.e., BACH1), suggesting that heme may have evolved to allow for regulation of protein stability by modulating the dynamics of “substrate-ubiquitin ligase” interaction. In this context, our team is poised to address the fundamental question: how does heme control protein degradation? To answer this question, we investigate how UPS machineries exploit heme to control protein degradation. 

Understand how the heme-oxidative stress pathway regulates gene expression by modulating the activity of intracellular transcriptional sensors

Recent pioneering studies revealed how heme signaling regulates a number of pathways, ranging from mitochondrial respiration to metabolism and circadian rhythms, by reversibly binding to and modulating the activity of “so-called” heme-sensor proteins. Heme-sensors participate in diverse biological processes including protein translation and transcription. In particular, it has been shown that binding of heme to transcriptional regulators switches on/off their activity and modulates the transcription of various enzymes and proteins that are critical to cellular physiology.
Despite these groundbreaking studies have provided critical data on how heme influences gene expression via regulation of transcriptional sensors, to date there is a limited understanding of the molecular events and machineries allowing signaling heme to modulate the cellular transcriptome. To broaden our understanding of this biological process, our team focuses on:

1.  The mechanisms of regulation of the heme-sensor transcription factor BACH1.

This transcriptional regulator is a major molecular link between the cellular heme levels, the redox state, and the transcriptional response. BACH1 functions both as a transcriptional repressor and activator, and regulates different biological processes like metabolism, cell growth, and cell cycle progression. Experimental evidence suggests that this pleiotropic transcriptional behavior is achieved by establishing different protein-protein networks with other transcriptional regulators that modulate its activity. However, to date, little is known about the specific transcriptional co-regulators working in conjunction with BACH1 to control certain transcriptional programs in response to heme fluctuations. Knowledge of these specific protein complexes is key to our understanding of how heme modulates BACH1 functions in physiology and of the mechanisms by which KEAP1/NRF2 mutant cancer cells rewire the transcriptional output of BACH1 to promote malignancy. To identify BACH1’s transcriptional co-regulators, we integrate results from standard immuno-purification purifications and BioID-based proximity labeling of BACH1 followed by mass spectrometry analysis. We anticipate that the results of this work will shed light on the mechanisms that control the transcriptional programs regulated by heme-BACH1, and is likely to uncover new mechanistic links between mutations in the KEAP1/NRF2 pathway in lung cancer and activation of pro-tumorigenic functions of BACH1.

2.  The identification of new heme-sensor transcription factors which may lead us to exploring uncharted biological fields.

To discover these new regulators, we will combine proteomic and genetic strategies. Specifically, we will use proximity labeling approaches to identify transcriptional regulators interacting with heme. Moreover, we will integrate these analyses with genome-wide CRISPR screenings coupled with transcriptomic profiling techniques to identify the factors that implement the heme-controlled transcription.

Unravel the mechanisms linking alteration of the heme-oxidative stress pathway to lung tumor development and progression

In cancers, heme signaling can be deregulated through alteration of the oxidative stress homeostasis. Oxidative stress is induced by elevated intracellular levels of reactive oxygen species (ROS). ROS play pleiotropic roles in tumorigenesis; while ROS are pro-tumorigenic, high ROS levels are cytotoxic. Cancer cells exhibit aberrant redox homeostasis that allows them to thrive under conditions of high oxidative burden. Specifically, hyperproliferation of tumor cells is accompanied by high ROS production, however, cancer cells accommodate high ROS levels by increasing their antioxidant status to optimize ROS-driven proliferation, while at the same time avoiding ROS thresholds that would trigger senescence and apoptosis.
To maintain oxidative homeostasis and sustain tumor growth, ~30% of non-small cell lung cancers (NSCLCs) increase the transcription of antioxidant genes by acquiring either stabilizing mutations in the transcription factor NRF2 (the NFE2L2 gene product) or by selecting for Loss-of-Function (LOF) mutations in its negative regulator, KEAP1. NRF2 increases the cells antioxidant defense by upregulating, among the others, genes involved in glutathione and NADPH metabolism as well as genes involved in the maintenance of signaling heme homeostasis. Excess of signaling heme is cytotoxic since it catalyzes the formation of ROS through Fenton reaction, resulting in oxidative stress. In turn, oxidative stress elicits heme release from heme-binding proteins, thus amplifying heme toxicity. NRF2 avoids the self-amplifying, pro-oxidant effects of signaling heme by inducing the transcription of genes regulating heme homeostasis, among which heme-oxygenase-1 (HO-1), the enzyme degrading signaling heme.
 
Using CRISPR/Cas9 in KP (KRAS-G12D; p53-/-) GEMM (i.e., genetically engineered mouse model), we recently discovered that LOF mutations of KEAP1 in NSCLC promote tumor progression via alteration of the heme signaling. Our findings contributed to identify novel vulnerabilities in lung cancer patients harboring alteration of the KEAP1/NRF2 pathway, and demonstrated that drugs targeting the heme pathway can be effectively used as therapeutics to inhibit the progression of these cancers. However, given that the heme signaling potentially targets multiple cellular pathways, effectors and substrates, the role of heme pathway in these cancers remains to be defined.
Today, our laboratory aims to achieve a deep understanding of the mechanisms linking alterations of the heme signaling to cancer development, and, in particular, to KEAP1/NRF2 mutant lung tumors pathogenesis. Our ultimate goal is to identify genotype-specific cancer vulnerabilities which may pave the road for the design of new personalized genotype-based therapies. To pursue our goals, we focus on:
 
1.  Understanding how KEAP1/NRF2 mutations in lung cancer impact on the heme-UPS regulated protein degradation.

For our analyses, we use orthogonal approaches combining biochemistry, proteomics and mouse genetics to identify the heme-UPS substrates which are deregulated in cancer and to determine their role in the mechanisms of tumorigenesis. 
 
2.  Investigating how alteration of heme-BACH1 impacts on the biology of KEAP1/NRF2 mutant lung cancer models harboring mutations in additional signaling pathways that frequently co-occur in patients.

To replicate these specific lung cancer patient genotypes, we use CRISPR/Cas9. Currently, we are generating lung cancer models harboring mutations in the oncogene KRAS and on the tumor suppressor LKB1. These two genes are mutated in ~30% (for KRAS) and ~45% (for LKB1) of the lung cancer patients with KEAP1 lesions, respectively. We use genomic approaches to identify the targets of the heme-BACH1 pathway in these tumor models, and molecular biology together with mouse genetics to understand how alteration of these targets influence tumor evolution.